Diketopiperazine salts for drug delivery and related methods

ABSTRACT

Drug delivery systems have been developed based on the formation of diketopiperazine carboxylate salts and microparticles containing the same. The systems may further comprise a bioactive agent. Related methods for making and using the biologically active agent delivery compositions are also provided. In certain embodiments, the pharmaceutically acceptable salts described can be formed by removal of solvent by methods including distillation, evaporation, spray drying or lyophilization.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/150,474, filed Jan. 8, 2014, which is a continuation of U.S.patent application Ser. No. 13/592,142 (now U.S. Pat. No. 8,653,085),filed Aug. 22, 2012, which is a divisional of U.S. patent applicationSer. No. 12/886,226 (now U.S. Pat. No. 8,278,308), filed Sep. 20, 2010,which is a divisional of U.S. patent application Ser. No. 11/210,710(now U.S. Pat. No. 7,820,676), filed Aug. 23, 2005, which claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationNo. 60/603,761 filed Aug. 23, 2004. The entire contents of each of theseapplications are incorporated by reference herein.

FIELD

This invention is generally in the field of drug delivery related toboth small molecule and macromolecular drugs. More particularly it isrelated to 2,5-diketopiperazine salts, their use in the formulation ofsuch drugs including therapeutic, prophylactic and diagnostic agents,stabilizing agents and systems for their delivery.

BACKGROUND TO THE INVENTION

Drug delivery has been a persistent challenge in the pharmaceuticalarts, particularly when a drug is unstable and/or poorly absorbed at thelocus in the body to which it is administered. One such class of drugsincludes 2,5-diketopiperazines, which is represented by the compound ofthe general Formula 1 as shown below where E=N.

These 2,5 diketopiperazines have been shown to be useful in drugdelivery, particularly those bearing acidic R groups (see for exampleU.S. Pat. No. 5,352,461 entitled “Self Assembling Diketopiperazine DrugDelivery System;” U.S. Pat. No. 5,503,852 entitled “Method For MakingSelf-Assembling Diketopiperazine Drug Delivery System;” U.S. Pat. No.6,071,497 entitled “Microparticles For Lung Delivery ComprisingDiketopiperazine;” and U.S. Pat. No. 6,331,318 entitled“Carbon-Substituted Diketopiperazine Delivery System,” each of which isincorporated herein by reference in its entirety for all that it teachesregarding diketopiperazines and diketopiperazine-mediated drugdelivery). Diketopiperazines can be formed into particles thatincorporate a drug or particles onto which a drug can be adsorbed. Thecombination of a drug and a diketopiperazine can impart improved drugstability. These particles can be administered by various routes ofadministration. As dry powders these particles can be delivered byinhalation to specific areas of the respiratory system, depending onparticle size. Additionally, the particles can be made small enough forincorporation into an intravenous suspension dosage form. Oral deliveryis also possible with the particles incorporated into a suspension,tablets or capsules; or dissolved in an appropriate solvent.Diketopiperazines may also facilitate absorption of an associated drug.Nonetheless difficulties can arise when diketopiperazines are diacids,or are in diacid form(s), due to the limited solubility of these diacidsat non-basic pH (i.e., neutral or acid pH). Another difficulty arisesbecause these diacid diketopiperazines may form disadvantageousassociation(s) with some drugs.

Therefore there is a need for diketopiperazine compositions havinggreater solubility at a neutral and/or acidic pH and methods for theiruse in the manufacture of therapeutic compositions.

SUMMARY OF THE INVENTION

The present invention provides improved drug delivery systems comprisingcarboxylate salts of heterocyclic compounds in combination with one ormore drugs. In one embodiment of the present invention the heterocycliccompounds form microparticles that incorporate the drug or drugs to bedelivered. These microparticles include microcapsules, which have anouter shell composed of either the heterocyclic compound alone or incombination with one or more drugs. The heterocyclic compounds of thepresent invention include, without limitation, diketopiperazines,diketomorpholines and diketodioxanes and their substitution analogs. Theheterocyclic compositions of the present invention comprise rigidhexagonal rings with opposing heteroatoms and unbonded electron pairs.

Specifically preferred embodiments include, without limitation,derivatives of 3,6-di(4-aminobutyl)-2,5-diketopiperazine, such as3,6-di(succinyl-4-aminobutyl)-2,5-diketopiperazine,3,6-di(maleyl-4-aminobutyl)-2,5-diketopiperazine, 3,6-di(citraconyl-4-aminobutyl)-2,5-diketopiperazine, 3,6-di(glutaryl-4-aminobutyl)-2,5-diketopiperazine, 3,6-di(malonyl-4-aminobutyl)-2,5-diketopiperazine, 3,6-di(oxalyl-4-aminobutyl)-2,5-diketopiperazine, and3,6-di(fumaryl-4-aminobutyl)-2,5-diketopiperazine (hereinafter fumaryldiketopiperazine or FDKP). Additionally, nonsymmetrical derivatives ofthe aforementioned are also contemplated. However, it is specificallynoted herein that lithium salts of 2,5-diaspartyl-3,6-diketopiperazineand 2,5-diglutamyl-3,6-diketopiperazine (as defined further below) arenot considered within the scope of the present invention and as such arehereby specifically disclaimed.

Representative drugs useful with the drug delivery systems of thepresent invention include, without limitation, insulin and otherhormones, peptides, proteins, polysaccharides, such as heparin, nucleicacids (such as plasmids, oligonucleotides, antisense, or siRNA), lipidsand lipopolysaccharides, anticoagulants, cytotoxic agents, antigens andantibodies and organic molecules having biological activity such as manyof the antibiotics, anti-inflammatories, antivirals, vaso- andneuroactive agents.

In one embodiment of the present invention, apharmaceutically-acceptable salt of a heterocyclic compound is providedaccording to Formula 1:

wherein R₁ or R₂ comprise at least one carboxylate functional group, E₁and E₂ comprise N or O and the salt further comprises at least onecation. In another embodiment, the heterocyclic compound comprises adiketopiperazine. In yet another embodiment, the carboxylate group isterminally located. In another embodiment of the pharmaceuticallyacceptable salt, R₁ and R₂ comprise 4-X-aminobutyl and X is selectedfrom the group consisting of succinyl, glutaryl, maleyl and fumaryl. Instill another embodiment, the cation is selected from the groupconsisting of sodium, potassium, calcium, lithium, triethylamine,butylamine, diethanolamine and triethanolamine.

In another embodiment of the present invention, thepharmaceutically-acceptable salt is not a lithium salt of2,5-diaspartyl-3,6-diketopiperazine or2,5-diglutamyl-3,6-diketopiperazine.

In an embodiment of the present invention, a therapeutic composition isprovided comprising a pharmaceutically acceptable salt of a heterocycliccompound according to Formula 1, wherein R₁ or R₂ comprise at least onecarboxylate functional group; E₁ and E₂ comprise N or O; the saltfurther comprises at least one cation; and the composition furthercomprises a biologically active agent. Biologically active agentssuitable for inclusion in the compositions of the present inventioninclude hormones, anticoagulants, immunomodulating agents, cytotoxicagents, antibiotics, antivirals, antisense, antigens, antibodies andactive fragments and analogues thereof. In one embodiment thebiologically active agent is insulin.

In another embodiment, the therapeutic composition of the presentinvention is formulated in a liquid such as a solution or a suspension.

In yet another embodiment, the therapeutic composition of the presentinvention is a precipitate and the precipitate is formulated into asolid dosage form suitable for oral, buccal, rectal, or vaginaladministration. The solid dosage form may be a capsule, a tablet, and asuppository.

In an embodiment, the therapeutic composition of the present inventionis a dry powder and the particles of said dry powder have a diameterbetween about 0.5 microns and 10 microns. In one aspect of theembodiment the dry powder is suitable for pulmonary administration.

In another embodiment of the present invention, a method of preparing asolid composition for drug delivery is provided comprising: preparing asolution containing a biologically active agent and apharmaceutically-acceptable salt of a heterocyclic compound in a solventand removing the solvent by a method selected from the group consistingof distillation, evaporation, and lyophilization. In one embodiment, thepharmaceutically-acceptable salt of a heterocyclic compound has thestructure according to Formula 1 wherein R₁ or R₂ comprise at least onecarboxylate functional group, E₁ and E₂ comprise N or O, and the saltfurther comprises at least one cation.

In yet another embodiment of the present invention, the method ofpreparing a solid composition for drug delivery further comprises thestep of micronizing the solid to form a dry powder.

In an embodiment of the present invention, a method of preparing a drypowder for drug delivery is provided comprising spray drying a solutionof a pharmaceutically acceptable salt of a heterocyclic compound and abiologically active agent to form a dry powder wherein the dry powderreleases a biologically active agent. In one embodiment, thepharmaceutically-acceptable salt of a heterocyclic compound has thestructure according to Formula 1 wherein R₁ or R₂ comprise at least onecarboxylate functional group, E₁ and E₂ comprise N or O, and the saltfurther comprises at least one cation. In another embodiment, theparticles of the dry powder are suitable for pulmonary delivery. In yetanother embodiment, the particles of the dry powder have a rugosity ofless than 2.

In an embodiment of the present invention, a composition for deliveringbiologically active agents is provided wherein the composition comprisesa pharmaceutically acceptable salt of a heterocyclic compound and abiologically active agent spray dried to form a dry powder such that thedry powder releases said biologically active agents. In one embodiment,the pharmaceutically-acceptable salt of a heterocyclic compound has thestructure according to Formula 1 wherein R₁ or R₂ comprise at least onecarboxylate functional group, E₁ and E₂ comprise N or O, and the saltfurther comprises at least one cation. In another embodiment, theparticles of the dry powder are suitable for pulmonary delivery. In yetanother embodiment, the particles of the dry powder have a rugosity ofless than 2.

In another embodiment of the present invention, a microparticulatesystem for drug delivery is provided comprising a composition ofpharmaceutically acceptable salt of a heterocyclic compound and abiologically active agent and wherein the composition releases abiologically active agent. In one embodiment, thepharmaceutically-acceptable salt of a heterocyclic compound has thestructure according to Formula 1 wherein R₁ or R₂ comprise at least onecarboxylate functional group, E₁ and E₂ comprise N or O, and the saltfurther comprises at least one cation. The biologically active agent caninclude hormones, anticoagulants, immunomodulating agents, cytotoxicagents, antibiotics, antivirals, antisense, antigens, antibodies andactive fragments and analogues thereof.

In yet another embodiment of the present invention, the composition ofthe microparticulate system is a dry powder which releases abiologically active agent in the pulmonary system. The composition canfurther be delivered to the pulmonary system. The composition of themicroparticulate system can be absorbed into the systemic bloodcirculation or act locally in the lung after delivery to the pulmonarysystem.

In an embodiment of the present invention, the composition of themicroparticulate system comprises a liquid for drug delivery and whereinthe absorption of the biologically active agent is facilitated by thediketopiperazine. In one embodiment the liquid is administered orally.

In another embodiment of the present invention, the composition of themicroparticulate system comprises a precipitate and wherein theabsorption of the biologically active agent is facilitated by thediketopiperazine. In one embodiment the precipitate is administeredorally.

In an embodiment of the present invention, a method for delivery ofparticles to the pulmonary system is provided comprising: administeringvia inhalation to a patient in need of treatment an effective amount ofa biologically active agent in the form of a dry powder, the dry powderprepared by spray drying a solution comprising a composition of apharmaceutically acceptable salt of a heterocyclic compound and abiologically active agent, wherein the dry powder releases thebiologically active agent in the pulmonary system. In one embodiment,the pharmaceutically-acceptable salt of a heterocyclic compound has thestructure according to Formula 1 wherein R₁ or R₂ comprise at least onecarboxylate functional group, E₁ and E₂ comprise N or O, and the saltfurther comprises at least one cation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict a laser diffraction particle size analysis ofparticles made using a fumaryl dikopiperazine (FDKP) disodium saltaccording to one aspect of the present invention. (A) preparation A; (B)preparation B.

FIG. 2 depicts particle size determination by laser diffraction of aformulation of a FDKP disodium salt containing 25% insulin (w:w) madeaccording to the teachings of the present invention.

FIG. 3 depicts scanning electron microscopy (SEM) of a spray driedmicroparticle preparation of a FDKP disodium salt containing 25% insulin(w:w) made according to the teachings of the present invention.

FIG. 4 depicts an accelerated stability study of spray driedmicroparticles of a FDKP disodium salt/insulin formulation containing25% insulin made according to the teachings of the present invention(stippled) compared to control lyophilized powder (hatched).

FIG. 5 depicts the effect of solution concentration on insulin stabilityof spray dried microparticles of a FDKP disodium salt/insulinformulation containing 25% insulin made according to the teachings ofthe present invention compared to control lyophilized powder.

FIGS. 6A, 6B, 6C, and 6D depict SEM analysis of the insulin/disodiumFDKP salt microparticles formed by the solvent/anti-solventprecipitation according to the teachings of the present invention. FIG.6A (10 k x) and FIG. 6B (20K x) are in the 1 to 5 micron range while atlower magnification (FIG. 6C, 2.5 k x and FIG. 6D, 1.0 k x) particles inthe 10 to 40 micron range are seen.

FIG. 7 depicts particle size determination by laser diffraction of spraydried microparticles of a FDKP diammonium salt/insulin formulationcontaining 25% insulin (w:w) made according to the teachings of thepresent invention.

FIG. 8 depicts particle size determination by laser diffraction of spraydried microparticles of a FDKP diammonium salt/insulin formulationcontaining 50% insulin (w:w) made according to the teachings of thepresent invention.

FIG. 9 depicts particle size determination by laser diffraction of spraydried microparticles of a diammonium salt of succinyl diketopiperazine(SDKP) containing 25% insulin (w:w) made according to the teachings ofthe present invention.

FIG. 10 depicts SEM of the FDKP ammonium salt formulated with 25%insulin according to the teachings of the present invention.

FIG. 11 depicts SEM of the SDKP ammonium salt formulated with 25%insulin according to the teachings of the present invention.

FIG. 12 depicts an accelerated stability study of the spray driedmicroparticles of a FDKP diammonium salt/insulin formulation containing25% or 50% insulin made according to the teachings of the presentinvention compared to control lyophilized powder.

FIG. 13 depicts the generation of the A₂₁ degradant during anaccelerated stability study of the spray dried microparticles of a FDKPdiammonium salt/insulin formulation containing 25% or 50% insulin madeaccording to the teachings of the present invention compared to controllyophilized powder.

FIG. 14 depicts an accelerated stability study of the spray driedmicroparticles of a diammonium SDKP salt/insulin formulation containing25% insulin made according to the teachings of the present inventioncompared to control lyophilized powder.

FIG. 15 depicts the generation of the A₂₁ degradant during anaccelerated stability study of the spray dried microparticles of adiammonium SDKP salt/insulin formulation containing 25% insulin madeaccording to the teachings of the present invention compared to controllyophilized powder.

FIG. 16 depicts the aerodynamic performance of spray dried FDKP disodiumsalt/insulin particles containing increasing insulin concentrations madeaccording to the teachings of the present invention.

FIG. 17 depicts the aerodynamic performance of spray dried FDKPdiammonium salt/insulin particles containing increasing insulinconcentrations made according to the teachings of the present invention.

DEFINITION OF TERMS

Prior to setting forth the invention, it may be helpful to provide anunderstanding of certain terms that will be used hereinafter:

Acidic: As used herein, “acidic” refers to a pH range of from 0, up to,but not including 6.

Basic: As used herein, “basic” refers to a pH range of from 8, up to andincluding 14.

Biological agents: See “Drug” below.

Cargo: See “Drug” below.

Diketopiperazine: As used herein, “diketopiperazines” or “DKP” includesdiketopiperazines and derivatives and modifications thereof fallingwithin the scope of Formula 1.

Drug: As used herein, “drug”, “cargo” or “biological agent” refer to thepharmacologically active agent incorporated with the microparticlesdiscussed herein. Examples include proteins and peptides (whereinprotein is defined as consisting of 100 amino acid residues or more anda peptide is less than 100 amino acid residues), such as insulin andother hormones; polysaccharides, such as heparin; nucleic acids, such asplasmids, oligonucleotides, antisense, or siRNA; lipids andlipopolysaccharides; and organic molecules having biological activitysuch as many of the antibiotics, anti-inflammatories, antivitals, vaso-and neuroactive agents. Specific examples include hormones,anticoagulants, immunomodulating agents, cytotoxic agents, antibiotics,antivirals, antisense, antigens, and antibodies.

Dry powder: As used herein “dry powder” refers to a fine particulatecomposition that is not suspended or dissolved in a propellant, carrier,or other liquid. It is not meant to imply a complete absence of allwater molecules.

Microparticles: As used herein, the term “microparticles” includesmicrocapsules having an outer shell composed of either adiketopiperazine alone or a combination of a diketopiperazine and one ormore drugs. It also includes microspheres containing drug dispersedthroughout the sphere; particles of irregular shape; and particles inwhich the drug is coated in the surface(s) of the particle or fillsvoids therein.

Neutral: As used herein, “neutral” refers to a pH range of from 6, upto, but not including 8.

Weakly alkaline: As used herein, “weakly alkaline” refers to a pH rangeof from 8, up to, but not including 10.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved drug delivery systems comprisingcarboxylate salts of heterocyclic compounds in combination with one ormore drugs. In one embodiment of the present invention the heterocycliccompounds form microparticles that incorporate the drug or drugs to bedelivered. These microparticles include microcapsules, which have anouter shell composed of either the heterocyclic compound alone or incombination with one or more drugs. The heterocyclic compounds of thepresent invention include, without limitation, diketopiperazines,diketomorpholines and diketodioxanes and their substitution analogs. Theheterocyclic compositions of the present invention comprise rigidhexagonal rings with opposing heteroatoms and unbonded electron pairs.

One aspect of the present invention includes a drug delivery systemcomprising the carboxylate salts of heterocyclic compounds incombination with one or more drugs. In one embodiment of the presentinvention the heterocyclic compounds form microparticles thatincorporate the drug or drugs to be delivered. These microparticlesinclude microcapsules, which have an outer shell composed of either theheterocyclic compound alone or in combination with one or more drug(s).This outer shell may surround a core material. This outer shell may alsosurround or constitute microspheres that are either solid or hollow, ora combination thereof, which contain one or more drugs dispersedthroughout the sphere and/or adsorbed onto the surface of the sphere.The outer shell also may surround microparticles having irregular shape,either alone or in combination with the aforementioned microspheres.

In a preferred embodiment for pulmonary delivery, the microparticles arefrom about 0.1 microns to about ten microns in diameter. Within drugdelivery systems, these microparticles exhibit desirable sizedistributions as well as good cargo tolerance.

The heterocyclic compounds of the present invention include, withoutlimitation, diketopiperazines, diketomorpholines and diketodioxanes andtheir substitution analogs. These heterocyclic compositions compriserigid hexagonal rings with opposing heteroatoms and unbonded electronpairs. The general formula for diketopiperazine and its analogs is shownbelow in the compound of Formula 1.

In the compound of Formula 1 the ring atoms E₁ and E₂ at positions 1 and4 are either O or N. At least one of the side-chains R₁ and R₂ locatedat positions 3 and 6 respectively contains a carboxylate group (i.e.,OR). In one embodiment of the present invention these carboxylate groupsare located along the side chains (R₁ and/or R₂) as pendent groups, inanother embodiment the carboxylate is located intra-chain (an ester) andyet in another embodiment the carboxylate groups are terminal.

General methods for the synthesis of diketopiperazines are known in theart and have been described in U.S. Pat. Nos. 5,352,461, 5,503,852, and6,331,318 which have been cited and incorporated herein by referenceabove. In a preferred embodiment of the invention the diketopiperazineis a derivative of 3,6-di(4-aminobutyl)-2,5-diketopiperazine, which maybe formed by condensation of the amino acid lysine. Exemplaryderivatives include 3,6-di(succinyl-4-aminobutyl)-(succinyldiketopiperazine or SDKP), 3,6-di(maleyl-4-aminobutyl)-,3,6-di(citraconyl-4-aminobutyl)-, 3,6-di(glutaryl-4-aminobutyl)-,3,6-di(malonyl-4-aminobutyl)-, 3,6-di(oxalyl-4-am inobutyl)-, and3,6-di(fumaryl-4-aminobutyl)-2,5-diketopiperazine (hereinafter fumaryldiketopiperazine or FDKP). Additionally, nonsymmetrical derivatives ofthe aforementioned compounds are also contemplated. However, it isspecifically noted herein that the lithium salts of2,5-diaspartyl-3,6-diketopiperazine and2,5-diglutamyl-3,6-diketopiperazine are not considered within the scopeof the present invention and as such are hereby specifically disclaimed.The free acids of these disclaimed compounds are depicted below inFormula 2 and Formula 3 respectively.

For convenience, the compound of Formula 2 will be referred tohereinafter as 2,5-diaspartyl-3,6-diketopiperazine. The compound ofFormula 3 will be referred to hereinafter as2,5-diglutamyl-3,6-diketopiperazine. It is understood that all otherheterocyclic compounds based on Formula 1 are considered within thescope of the present invention.

For exemplary purposes, diketopiperazines salts and their derivativeswill be described in detail. These compounds are the preferredembodiments of the present invention. However, this does not excludeother heterocyclic compounds based on the compound of Formula 1.

The use of DKP salts for the delivery of phosphodiesterase type5-inhibitors is described in co-pending U.S. patent application Ser. No.11/210,709 filed Aug. 23, 2005 and entitled “Pulmonary Delivery ofInhibitors of Phosphodiesterase Type 5” and known to all by U.S.Provisional Patent Application No. 60/603,764, which is herebyincorporated by reference in its entirety. Pulmonary drug delivery usingDKP microparticles is disclosed in U.S. Pat. No. 6,428,771 entitled“Method For Drug Delivery To The Pulmonary System”, which is herebyincorporated by reference in its entirety.

Diketopiperazine facilitate transcellular transport of biologicallyactive agents across biological tissues however they are not penetrationenhancers. Penetration enhancers are compounds that improve drugmovement across biological tissues by disrupting cell membranes.Examples of penetration enhancers are surfactants and soaps.Diketopiperazines do not disrupt cell membranes either in vitro or invivo. In vitro studies demonstrate that FDKP does not disrupt cellmembranes or tight junctions and does not compromise cell viability.Diketopiperazine/insulin powder compositions are soluble at thephysiological pH of the lung surface and dissolve rapidly afterinhalation. Once dissolved, the DKP facilitates passive transcellulartransport of the insulin.

Applicants have discovered improved diketopiperazine compositions havinggreater solubility at a neutral and/or acidic pH. Applicants have alsodiscovered that therapeutic complexes between improved diketopiperazinesand drug(s) of interest can be formed.

The salts of the present invention can be prepared by reacting thediketopiperazine free acid with a solution of an appropriate base asdescribed in Examples 1 and 2 below. In a preferred embodiment, the saltis a pharmaceutically acceptable salt such as the sodium (Na), potassium(K), lithium (Li), magnesium (Mg), calcium (Ca), ammonium, or mono-, di-or tri-alkylammonium (as derived from triethylamine, butylamine,diethanolamine, triethanolamine, or pyridines, and the like) salts ofdiketopiperazine, for example. The salt may be a mono-, di-, or mixedsalt. Higher order salts are also contemplated for diketopiperazines inwhich the R groups contain more than one acid group. In other aspects ofthe invention, a basic form of the agent may be mixed with the DKP inorder to form a drug salt of the DKP, such that the drug is the countercation of the DKP.

For drug delivery, biologically active agents or drugs havingtherapeutic, prophylactic, or diagnostic activities can be deliveredusing diketopiperazines. Essentially, the biologically active agent isassociated with the diketopiperazine particles of the present invention.As used herein, “associated” means a biologically activeagent-diketopiperazine composition formed by, among other methods,co-precipitation, spray drying or binding (complexation) of thediketopiperazine with the biologically active agent. The resultingdiketopiperazine particles include those that have entrapped,encapsulated and/or been coated with the biologically active agent.While the exact mechanism of association has not been conclusivelyidentified, it is believed that the association is a function ofphysical entrapment (molecular entanglement) in addition toelectrostatic attraction including hydrogen bonding, van der Waal'sforces and adsorption.

The biologically active agents that can be associated with thediketopiperazine particles of the present invention include, but are notlimited to, organic or inorganic compounds, proteins, or a wide varietyof other compounds, including nutritional agents such as vitamins,minerals, amino acids, carbohydrates, sugars, and fats. In preferredembodiments, the drugs include biologically active agents that are to bereleased in the circulatory system after transport from the GI tractfollowing oral delivery. In other preferred embodiments the materialsare biologically active agents that are to be released in thecirculatory system following pulmonary or nasal delivery. In otherpreferred embodiments the materials are biologically active agents thatare to be release in the central nervous system following nasaldelivery. Additionally, the drug can be absorbed through mucosal tissuesuch as rectal, vaginal, and/or buccal tissue. Non-limiting examples ofbiologically active agents include proteins and peptides (whereinprotein is defined as consisting of 100 amino acid residues or more anda peptide is less than 100 amino acid residues), such as insulin andother hormones, polysaccharides, such as heparin, nucleic acids (such asplasmids, oligonucleotides, antisense, or siRNA), lipids andlipopolysaccharides, and organic molecules having biological activitysuch as many of the antibiotics, anti-inflammatories, vasoactive agents(including agents used to treat erectile dysfunction) and neuroactiveagents. Specific non-limiting examples include steroids, hormones,decongestants, anticoagulants, immunomodulating agents, cytotoxicagents, antibiotics, antivirals, anesthetics, sedatives,antidepressants, cannabinoids, anticoagulants, antisense agents,antigens, and antibodies. In some instances, the proteins may beantibodies or antigens which otherwise would have to be administered byinjection to elicit an appropriate response. More particularly,compounds that can be associated with the diketopiperazine compositionsof the present invention include insulin, heparins, calcitonin,felbamate, parathyroid hormone and fragments thereof, growth hormone,erythropoietin, glucagon-like peptide-1, somatotrophin-releasinghormone, follicle stimulating hormone, cromolyn, adiponectin, RNAse,ghrelin, zidovudine, didanosine, tetrahydrocannabinol (i.e.,cannabinoids), atropine, granulocytes colony stimulating factor,lamotrigine, chorionic gonadotropin releasing factor, luteinizingreleasing hormone, beta-galactosidase and Argatroban. Compounds with awide range of molecular weight can be associated, for example, between100 and 500,000 grams per mole.

Imaging agents including metals, radioactive isotopes, radiopaqueagents, and radiolucent agents, can also be incorporated intodiketopiperazine delivery systems. Radioisotopes and radiopaque agentsinclude gallium, technetium, indium, strontium, iodine, barium, andphosphorus.

Additionally the drugs can be in various forms, such as unchargedmolecules, metal or organic salts, or prodrugs. For acidic drugs, metalsalts, amines or organic cations (e.g., quaternary ammonium) can in somecases be used.

In some embodiment, the drugs include biologically active agents thatare to be released in the circulatory system after transport from thegastrointestinal tract following oral delivery. In other embodiments,the biologically active agents are to be released in the circulatorysystem following pulmonary or nasal delivery. In still otherembodiments, the biologically active agents are to be released in thecentral nervous system following nasal delivery. Additional, the drugscan be absorbed through mucosal tissue such as rectal, vaginal, and/orbuccal tissue.

Some of these biological agents are unstable in gastric acid, diffuseslowly through gastrointestinal membranes, are poorly soluble atphysiological pH, and/or are susceptible to enzymatic destruction in thegastrointestinal tract. The biological agents are combined with thediketopiperazine salts to protect them in the gastrointestinal tractprior to release in the blood stream. In a preferred embodiment thediketopiperazines are not biologically active and do not alter thepharmacologic properties of the therapeutic agents.

To associate one or more drugs with a DKP salt, the drug and the DKPsalt are preferably mixed in solution or suspension and subsequentlydried. Either component may be present as solute or suspendate. Indifferent embodiments the mixture is spray dried or lyophilized.

Spray drying is a thermal processing method used to form, load or dryparticulate solids from a variety of solutions or suspensions. The useof spray drying for the formation of dry particulate pharmaceuticals isknown in the art however in the past its use had been limited by itsincompatibility with biological macromolecular drugs, including protein,peptides and nucleic acids due to the nature of the spray dryingprocess. During spray drying, a solution or suspension is formed intodroplets through aerosolization and then passed through a heated gasstream having sufficient heat energy to evaporate water and solvents inthe particles to a desired level before the particles are collected. Theinlet temperature is the temperature of the gas stream leaving itssource and its level is selected based upon the lability of themacromolecule being treated. The outlet temperature is a function of theinlet temperature, the heat load required to dry the product along withother factors.

The present inventors have unexpectedly determined that the particles ofthe present invention, have aerodynamic performance which improves withincreasing content of a biologically active agent which has not beenseen with other particles. The respirable fraction (% rf), thepercentage of particles between 0.5 and 5.8 microns in diameter, of thespray dried particles of the present invention increases with increasinginsulin content, rather than decreasing as was expected. Therefore usingthe methods of the present invention, diketopiperazine microparticlescan be formed which have higher biologically active agent content thatwas previously achievable.

Additionally, the present inventors have surprisingly determined thatspray dried FDKP disodium salt/insulin compositions have increasedinsulin stability as the concentration of the FDKP disodium salt in thestarting solution increases. Stability was measured by insulin lossafter 17 days at 40° C./75% relative humidity. For example, 8.5% insulinwas lost from powder spray dried from a solution containing 37 mg/mLsolids (total weight of FDKP disodium salt/insulin). By comparison, 4.5%insulin was lost from powder spray dried from a solution containing 45mg/mL solids and 2.7% insulin was lost from powder spray dried from asolution containing 67 mg/mL solids.

In a further observation, inlet temperature was found to have surprisingeffects on insulin stability. The data indicate that insulin stabilityin the powder increases with increasing inlet temperature as measured byinsulin loss after 17 days at 40°/75% RH. For example, about 4% insulinwas lost from powder spray dried at an inlet temperature of 180° C. Bycomparison, <1% insulin was lost from powder spray dried at an inlettemperature of 200° C.

In an embodiment of the present invention, microparticles suitable fordelivery to the pulmonary system are provided wherein the microparticleshave a rugosity of less than 2. Another aspect of the present inventioninfluenced by spray drying is the particle morphology, measured byrugosity, which the ratio of the specific area and the surface areacalculated from the particle size distribution and particle density. Thedrying operation may be controlled to provide dried particles havingparticular characteristics, such as rugosity. Rugosity of spray driedparticles is a measure of the morphology of the surface of theparticles, such as the degree of folding or convolution.

It had previously been thought that a rugosity above 2 was needed inorder to obtain particles with sufficient dispersability to form afree-flowing powder. Surprisingly, the present inventors have producedparticles suitable for pulmonary delivery with a rugosity below 2

The microparticle formulations of the present invention can beadministered as a liquid or solid form. These can include solutions,suspensions, dry powders, tablets, capsules, suppositories, patches fortransdermal delivery, and the like. These different forms offerdistinct, but overlapping, advantages. The solid forms provideconvenient bulk transport of drugs and can improve their stability. Theycan also be formed into microparticles enabling administration byinhalation specifically to the nasal mucosa or deep lung, depending onthe size of the microparticle. Diketopiperazines can also facilitateabsorption of the associated drug even when delivered as a solution.Some of the DKP salts (for example, the sodium and potassium salts)offer improved solubility at neutral and acidic pH as compared to thefree acid, which can lead to improved absorption in the stomach oforally administered solid forms.

Dikeopiperazine salt counter cations may be selected to produce saltshaving varying solubilities. These varying solubilities can be theresult of differences in dissolution rate and/or intrinsic solubility.By controlling the rate of DKP salt dissolution, the rate of drugabsorption from the DKP salt/drug combination can also be controlled toprovide formulations having immediate and/or sustained release profiles.For example, sodium salts of organic compounds are characteristicallyhighly soluble in biological systems, while calcium salts arecharacteristically only slightly soluble in biological systems. Thus, aformulation comprised of a DKP sodium salt/drug combination wouldprovide immediate drug absorption, while a formulation comprised of aDKP calcium salt/drug combination would provide slower drug absorption.A formulation containing a combination of both of the latterformulations could be used to provide immediate drug absorption followedby a period of sustained absorption.

Diketopiperazine salt formulations of biologically active agents may beadministered orally. Microparticles, depending on the chemical natureand size, are absorbed through the epithelial lining of thegastrointestinal tract into the bloodstream or lymphatic system.Alternatively, the composition can be administered as a solution inwhich the DKP salt serves to facilitate the absorption of the drug.Additionally, the microparticles can be administered as a suspension ora solid dosage form that dissolves completely and is absorbed followingdissolution.

For parenteral administration, microparticles of less than five micronsreadily pass through a needle for intravenous administration. Suitablepharmaceutical carriers, for example, phosphate buffered saline, areknown and commercially available. Similarly, microparticles can beinjected or implanted subcutaneously, intramuscularly, orintraperitoneally. Additionally, the microparticles can be placed in animplantable device to facilitate sustained and/or controlled delivery.

For topical or transdermal administration, microparticles can besuspended in a suitable pharmaceutical carrier for administration usingmethods appropriate for the carrier and site of administration. Forexample, microparticles are administered to the eye in a buffered salinesolution, at a pH of approximately 7.4, or in an ointment such asmineral oil. The dosage will be dependent on the compound to be releasedas well as the rate of release. The microparticles, or aggregations ofmicroparticles into films, disks, or tablets, with incorporated compoundcan be administered to the skin in an ointment, cream, or patch.Suitable pharmaceutical carriers are known to those skilled in the artand commercially available. Mucosal administration, including buccal,vaginal, rectal, nasal administration is also contemplated.

Pulmonary delivery can be very effectively accomplished using drypowders comprising the microparticles of the invention and can lead torapid absorption into the circulation (bloodstream). Dry powder inhalersare known in the art and particularly suitable inhaler systems aredescribed in U.S. patent application Ser. Nos. 09/621,092 and10/655,153, both entitled “Unit Dose Capsules and Dry Powder Inhaler”,which are hereby incorporated by reference in their entirety.Information on pulmonary delivery using microparticles comprisingdiketopiperazine can be found in U.S. Pat. No. 6,428,771 entitled“Method for Drug Delivery to the Pulmonary System,” which is herebyincorporated by reference in its entirety. The following examples aremeant to illustrate one or more embodiments of the invention and are notmeant to limit the invention to that which is described below.

EXAMPLES Example 1 Preparation A of FDKP Disodium Salt

Thirteen grams of fumaryl diketopiperazine (FDKP) (28.73 mmol, 1 equiv.)were placed into a 250 mL 3-neck round bottom flask equipped with areflux condenser, magnetic stir bar, and thermometer. The reaction wasrun under a nitrogen atmosphere. Water (150 mL) and 50% sodium hydroxide(4.48 g, 1.95 equiv.) were added sequentially to the flask. Theresulting yellow solution was heated to 50° C. and held for 2 hours. Thesolution was then hot filtered to remove any insoluble material. Thewater was removed from the sample via rotary evaporation. The recoveredsolids were dried in the vacuum oven (50° C., 30 inches of mercury)overnight. The salt was then assayed for moisture content (Karl Fischer)and sodium content (elemental analysis and titration). The yield of thesalt was from about 90% to about 95%.

Molecular Formula: C₂₀H₂₆N₄Na₂O₈.1.4809 H₂O

% Water by Karl Fischer titration: 5.1%

Elemental Analysis:

Calc C 45.92 H 5.58 N 10.71 Na 8.79 Found C 45.05 H 5.23 N 10.34 Na 9.18

Titration: 97% disodium salt (weight percent)

TABLE 1 Laser deffraction particle size analysis (Preparation Aparticles): Lot# X₁₀ X₁₆ X₅₀ X₈₄ X₉₀ X₉₉ VMD GSD Preparation A 1.60 μm1.44 μm 2.89 μm 4.60 μm 5.47 μm 19.20 μm 3.70 μm 1.59 Particle Size FineParticle Fraction Lot# <3 μm 0.5-5 μm (<5.8 μm) Preparation A 53.39%87.91% 91.46% VMD = Volume median diameter; GSD = geometric standarddeviation.

Example 2 Preparation B of FDKP Disodium Salt

Thirteen grams of FDKP (28.73 mmol, 1 equiv.) and ethanol (150 mL) wereplaced into a 250 mL 3-neck round bottom flask equipped with a refluxcondenser, magnetic stir bar, and thermometer. The reaction was rununder a nitrogen atmosphere. The slurry was heated to 50° C. Sodiumhydroxide, 50% w/w aqueous solution (4.71 g, 2.05 equiv.) was added inone portion. The resulting slurry was held at 50° C. for 2 hours. Thereaction contents were then cooled to ambient temperature (20-30° C.)and the solids isolated by vacuum filtration. The recovered salt waswashed with ethanol (300 mL) and acetone (150 mL) and dried in thevacuum oven (50° C., 30 inches of mercury) overnight. No furtherpurification was required. The salt was then assayed for moisturecontent (Karl Fischer) and sodium content (elemental analysis andtitration). The yield of the salt was from about 90% to about 95%.

Molecular Formula: C₂₀H₂₆N₄Na₂O₈.1.4503 H₂O

% Water by Karl Fischer titration: 5%

Elemental Analysis:

Calc C 45.97 H 5.57 N 10.72 Na 8.8 Found C 46.28 H 5.26 N 10.60 Na 8.96

Titration: 98.8% disodium salt (weight percent)

TABLE 2 Laser deffraction particle size analysis (Preparation Bparticles): Lot# X₁₀ X₁₆ X₅₀ X₈₄ X₉₀ X₉₉ VMD GSD Preparation A 1.55 μm1.36 μm 3.11 μm 5.53 μm 6.64 μm 14.04 μm 3.76 μm 1.75 Particle Size FineParticle Fraction Lot# <3 μm 0.5-5 μm (<5.8 μm) Preparation A 47.37%80.13% 86.01% VMD = Volume median diameter; GSD = geometric standarddeviation.

Example 3 Preparation A of FDKP Dilithium Salt

Ten grams of FDKP (22.10 mmol, 1 equiv.) and 100 mL of water were placedinto a 200 mL 3-neck round bottom flask equipped with a refluxcondenser, magnetic stir bar, and thermometer. The reaction was rununder a nitrogen atmosphere. In a separate flask, an aqueous solution oflithium hydroxide (1.81 g, 1.95 equiv.) in 40 mL of water was prepared.Once all of the lithium hydroxide had dissolved, this solution was addedin one portion to the aqueous slurry of FDKP. The resulting solution washeated to 50° C. and held for 1 hour. The reaction contents were thencooled to ambient temperature and filtered to remove any undissolvedparticles. The water was removed from the sample via rotary evaporation.The recovered solids were dried in a vacuum oven (50° C., 30 inches ofmercury) overnight. The salt was then assayed for moisture content (KarlFischer) and lithium content (elemental analysis and titration). Theyield of the salt was about 98%.

Molecular Formula: C₂₀H₂₆N₄Li₂O₈.0.0801 H₂O

Karl Fischer: 0.31%

Elemental Analysis:

Calc C 51.57 H 5.66 N 12.03 Li 2.98 Found C 50.98 H 5.74 N 11.95 Li 2.91

Titration: 98.3% dilithium salt (weight percent)

Example 4 Preparation A of FDKP Dipotassium Salt

Twelve grams of FDKP (26.52 mmol, 1 equiv.) were placed into a 250 mL3-neck round bottom flask equipped with a reflux condenser, magneticstir bar, and thermometer. The reaction was run under a nitrogenatmosphere. Potassium hydroxide (0.5N, 105 g, 1.98 equiv.) was added tothe flask. The resulting solution was heated to 50° C. and held for 2hours. The reactants were cooled to ambient temperature and the waterwas removed from the sample via rotary evaporation. The recovered solidswere dried in the vacuum oven (50° C., 30 inches of mercury) overnight.The salt was then assayed for moisture content (Karl Fischer) andpotassium content (elemental analysis and titration). The yield of thesalt was from about 95% to about 98%.

Molecular Formula: C₂₀H₂₆N₄K₂O₈.0.4529 H₂O

Karl Fischer: 4.98%

Elemental Analysis:

Calc C 44.75 H 5.05 N 10.44 K 14.56 Found C 44.88 H 4.74 N 10.36 K 14.34

Titration: 97.0% dipotassium salt (weight percent)

Example 5 Preparation B of FDKP Dipotassium Salt

Ten grams of FDKP (22.10 mmol, 1 equiv.) and ethanol (150 mL) wereplaced into a 250 mL 3-neck round bottom flask equipped with a refluxcondenser, magnetic stir bar, and thermometer. The reaction was rununder a nitrogen atmosphere. The slurry was heated to 50° C. Potassiumhydroxide (10N, 4.64 g, 2.10 equiv.) was added in one portion. Theresulting slurry was held at 50° C. for a minimum of 3 hours. Thereaction contents were cooled to ambient temperature (20-30° C.) and thesolids isolated by vacuum filtration. The recovered salt was washed withethanol (100 mL) and acetone (200 mL) and dried in a vacuum oven (50°C., 30 inches of mercury) overnight. No further purification wasrequired. The salt was then assayed for moisture content (Karl Fischer)and potassium content (elemental analysis and titration). The yield ofthe salt was from about 94% to about 98%.

Molecular Formula: C₂₀H₂₆N₄K₂O₆.0.6386 H₂O

Karl Fischer: 2.13%

Elemental Analysis:

Calc C 44.47 H 5.09 N 10.37 K 14.47 Found C 44.48 H 5.03 N 10.31 K 13.92

Titration: 97% dipotassium salt (weight percent)

Example 6 Preparation A of Disodium FDKP-Insulin Microparticles

Two and a half grams of FDKP disodium salt (Preparation A) was placed ina 250 mL beaker with a magnetic stir bar. The material was suspended in75 mL of deionized water. Insulin (0.84 g) was added to the FDKP saltsuspension. The resulting slurry was titrated to a pH of 8.3 with NH₄OHto form a solution. The FDKP disodium salt and insulin solution wasbrought to a volume of 100 mL with deionized water and filtered througha 0.22 μm polyethersulfone membrane. The solution was spray-dried usinga BUCHI® Mini Spray Dryer B-191 (Buchi Labortechnik AG, Switzerland)under the following conditions.

-   -   Inlet Temperature set at 170° C.    -   Outlet Temperature=75° C.    -   Aspiration rate 80% of maximum    -   Atomization=600 l/hr of dry nitrogen    -   Feed pump rate 25% of maximum (8.5 ml/min)    -   Nozzle chiller return water 22° C.

Example 7 Preparation B of Disodium FDKP-Insulin Microparticles

Five grams of FDKP disodium salt (Preparation B) was placed in a 250 mLbeaker with a magnetic stir bar. The material was suspended in 75 mL ofdeionized water. Insulin (1.68 g) was added to the FDKP salt suspension.The resulting slurry was titrated to a pH of 8.3 with NH₄OH to form asolution. The FDKP disodium salt and insulin solution was brought to avolume of 100 mL with deionized water and filtered through a 0.22 μmpolyethersulfone membrane. The solution was spray-dried using a BUCHI®Mini Spray Dryer B-191 (Buchi Labortechnik AG, Switzerland) under thefollowing conditions.

-   -   Inlet Temperature set at 149° C.    -   Outlet Temperature=75° C.    -   Aspiration rate 80% of maximum    -   Atomization=600 l/hr of dry nitrogen    -   Feed pump rate 25% of maximum (8.5 mL/min)    -   Nozzle chiller return water 23° C.

Example 8 Characterization of Disodium FDKP-Insulin Microparticles

The microparticles described in Examples 6 and 7 were subjected to laserdiffraction particle size analysis (SympatecGmbH, Germany) (FIGS. 1A and1B). The particles of Example 6 displayed an average respirable fraction(according to the USP definition of 0.5 to 5.8 microns) of 87.93% with astandard deviation of 1.60 and a % CV (coefficient of variation) of1.82. The particles of Example 7 displayed an average respirablefraction of 81.36% with a standard deviation of 4.20 and a % CV of 5.16.

Example 9 Pulmonary Administration of Disodium FDKP-Insulin

A dry powder containing the disodium FDKP salt and insulin is inhaled atthe beginning of meal. The particles that comprise the dry powder arepreferably in the range of approximately 0.5-5.8 microns in size. Theexact dosage is patient-specific, but generally on the order of 5-150Units of insulin per dose. The insulin absorption from this dosageregimen mimics physiologic first-phase insulin release, and attenuatespost-prandial blood glucose excursions.

Example 10 Preparation of an Oral Dosage Form

Spray-dried disodium FDKP/insulin powder as described in Examples 6 or 7is packed into hard gelatin capsules. The capsules can containapproximately 50-100 mg of powder. The FDKP salt/insulin powdersprepared in Examples 6 and 7 were 25% insulin by weight and insulinactivity was about 26 units/mg. Thus, 50 mg would be on the order of1300 units, significantly larger than a typical dose. About 2-30 mg ofthe FDKP salt/insulin powder is mixed with methyl cellulose (otherbulking agents are well known in the art) to make up the balance of thedesired mass.

Example 11 Oral Administration of Disodium FDKP-Insulin

Capsules containing the FDKP salt and insulin are taken before a meal.The exact dosage is patient-specific, but generally on the order ofapproximately 10-150 units of insulin is administered per dose. Thesubsequent insulin absorption attenuates post-prandial blood glucoseexcursions. This oral insulin formulation is used to replace pre-mealinsulin injections in patients with diabetes. Additionally, insulinabsorbed through the gastrointestinal tract mimics endogenous insulinsecretion. Endogenous insulin is secreted by the pancreas into theportal circulation. Insulin absorbed following oral administration alsogoes directly to the portal circulation. Thus, the oral route of insulinadministration delivers insulin to its site of action in the liver,offering the potential to control glucose levels while limiting systemicexposure to insulin. Oral insulin delivery using a combination ofinsulin and the diacid form of FDKP is hindered by the poor solubilityof the FDKP diacid in the low pH environment of the gastrointestinaltract. The FDKP salts, however, provide a local buffering effect thatfacilitates their dissolution in low pH.

Example 12 Preparation C of FDKP Di-Sodium Salt

Fifty grams of fumaryl diketopiperazine (FDKP, 221.01 mmol, 1 equiv.),water (200 mL), and 10 N sodium hydroxide (21.9 mL, 437.61 mmol, 1.98equiv.) were charged to a 1-liter, 4-neck, round bottom flask equippedwith a reflux condenser, overhead stirrer, nitrogen inlet, andthermometer. The mixture was heated to 50° C. to achieve a yellowsolution and ethanol (650 mL) was added over 15 minutes. When theaddition was complete, the slurry was held at 50° C. for 30-60 minutes.The reaction mixture was vacuum filtered and the isolated solids werewashed with ethanol (150 mL) and acetone (150 mL×2) then dried in avacuum oven (50° C., 30 inches of mercury) overnight. No furtherpurification was required. The salt was assayed for moisture content(Karl Fischer) and sodium content (elemental analysis and titration).The yield of the salt was from about 90% to about 95%.

Karl Fischer: 7.19%

Elemental Analysis:

Calc C 44.91 H 5.70 N 10.47 Na 8.6 Found C 45.29 H 5.47 N 10.59 Na 8.24

Titration: 98.8% disodium salt (weight percent)

The following are various processes described with regard to variousformulations of the present invention.

Example 13 FDKP Salt/Insulin Powder Prepared by Spray Drying

The disodium salt of FDKP (5 g) was dissolved in deionized water (150mL) and insulin (1.69 g) was added. The pH of the suspension wasadjusted to 8.3 with ammonium hydroxide (NH₄OH) to give a solution thatwas subsequently diluted to 200 mL with deionized water and filtered.The solution was spray dried using the following conditions:

-   -   Inlet temperature—200° C.    -   Outlet temperature—80° C.    -   Atomization gas—600 liter N₂/hr    -   Process gas—80% of maximum    -   The spray nozzle was cooled to 28° C.

The resultant particles were analyzed for their aerodynamic propertiesand the data are reported in Table 3.

TABLE 3 Aerodynamic properties of spray dried disodium FDKP/insulin.Sample % rf % empty % rf fill mmad gsd inlet ° C. % load LOD FDKPdisodium salt with 44.5 85.6 38.1 3.1 1.9 200 25.00 5.4 25% insulin(w:w)

Table 3 shows the respirable fraction (% rf), which is the percentage ofparticles between 0.5 and 5.8 microns in diameter, the percentage ofpowder that empties from the cartridge upon discharge (% empty), thepercentage of respirable fraction per fill (% rf fill, % rf X %empty—this measures the % of the respirable particles in the powderemptied from the cartridge, the mass median aerodymanic diameter (mmad),the inlet ° C. (the inlet temperature in degrees Celsius), thepercentage of load (% load—the insulin content of particles in weight%), and the loss on drying (LOD), a measure of the residual water in thepowder expressed as the % volatile material removed when the powder isdried in an oven overnight.

Particle size measured by laser diffraction demonstrated a size range ofapproximately 2 μm-15 μm and the data are displayed in Table 4 and inFIG. 2.

TABLE 4 Fine Particle Fraction Lot# Run X₁₀ X₅₀ X₉₀ VMD GSD (<5.8 μm)FDKP disodium salt with 168 2.14 μm 5.88 μm 15.16 μm 7.76 μm 2.10 49.21%25% insulin (w:w)

Scanning electron microscopy (SEM) was utilized to study particlemorphology. A representative SEM is shown in FIG. 3. The particlemorphology is consistent with a collapsed hollow sphere.

The stability of the disodium salt/insulin particles was evaluated underaccelerated room temperature conditions (40°/75% relative humidity[RH]). Compared to a control formulation prepared by lyophilization, thespray-dried particles demonstrated superior insulin stability asmeasured by insulin degradation (FIG. 4).

The starting concentration of the FDKP disodium salt/25% insulinsolution prior to spray drying was evaluated for its effect on finalparticle stability. The data (FIG. 5) shows that insulin stability onthe particle increases with increasing solution concentrations asmeasured by insulin loss after 17 days at 40°/75% RH.

Example 14 Solvent/Anti-Solvent Precipitation of a Solution of FDKPSalt/Insulin with an Organic Solvent

The precipitation was controlled using harmonic ultrasonic atomization.Alternate cavitation methods as well as high shear mixing andhomogenization are also applicable.

The disodium salt of FDKP (5 g) was dissolved in deionized water (80mL). Insulin (0.65 g) was added to the solution to produce a suspension.The pH of the suspension was adjusted to 8.3 with NH₄OH to obtain asolution that was diluted to 100 mL with deionized water and filtered.The particles were precipitated by pumping the insulin/disodium salt ofFDKP solution and ethanol in a 1:5 ratio through a duel inletatomization horn vibrating at a frequency between 20 kHz and 40 kHz. Theprecipitate was collected in a media bottle containing ethanol (200 mL).Post-precipitation the material was washed with ethanol and dried viarotary evaporation or by bubbling nitrogen through the suspension. Theparticles contained 12.5% insulin by weight. Particle morphology wasevaluated by SEM (FIGS. 6A, 6B, 6C, and 6D).

The particles illustrated in FIG. 6A (10 k x) and FIG. 6B (20K x) are inthe 1 to 5 micron range while at lower magnification (FIG. 6C, 2.5 k xand FIG. 6D, 1.0 k x) particles in the 10 to 40 micron range are seen.It is the non-binding hypothesis of the present inventors that thedrying methods utilized in this study resulted in recrystalization ofthe primary particles into much larger secondary particles and that theuse of a method that maintains a constant ratio of organic to aqueouscomponents throughout the drying process, such as spray drying, canpreserve the primary particles to the exclusion of the formation of asignificant number of secondary particles.

Example 15 In Situ Diammonium Salt Formation and Formulation

FDKP or SDKP (succinyl DKP) diammonium salt/insulin particles wereformed by spray drying. A representative procedure is given for the FDKPammonium salt/insulin formulation containing 25% insulin.

FDKP (5 g) was suspended in deionized water (150 mL) and titrated to apH of 7.5 to 8.0 with ammonium hydroxide (NH₄OH). Insulin (1.69 g) wasadded to the resulting solution (FDKP) to give a suspension. The pH ofthe suspension was adjusted to 8.3 with ammonium hydroxide (NH₄OH) togive a solution that was diluted to 200 mL with deionized water andfiltered. The powder was produced by spray drying the solution under thefollowing conditions.

-   -   Inlet temperature—200° C.    -   Outlet temperature—80° C.    -   Atomization gas—600 liter N₂/hr    -   Process gas—80% of maximum    -   The spray nozzle was cooled to 28° C.

The % rf of the diammonium salts is about 10% higher than the % rf ofthe disodium salt. The counter cation has a large effect on particleperformance. Also, the 50% FDKP ammonium salt/insulin powder has a % rfcomparable to that of the corresponding 25% FDKP ammonium salt/insulinpowder. This is surprising because with the powders prepared bylyophilization from the FDKP free acid, the % rf decreases as theinsulin content increases.

The resultant particles were analyzed for their aerodynamic propertiesand the data are reported in Table 5.

TABLE 5 Aerodynamic properties of spray dried diammonium FDKP/insulinand diammonium SDKP/insulin Sample % rf % empty % rf fill mmad gsd inlet° C. % load LOD FDKP diammonium salt 52.1 88.7 46.2 2.9 1.9 200 25.006.6 with 25% insulin (w:w) FDKP diammonium salt 55.7 85.4 47.5 2.9 1.8200 50.00 6.2 with 50% insulin (w:w) SDKP diammonium salt 56.0 90.1 55.73.0 2.0 200 25.00 3.8 with 25% insulin (w:w)

Particle size measured by laser diffraction and the data are displayedin Table 6 and in FIGS. 7-9.

TABLE 6 Fine Particle Fraction Lot# Run X₁₀ X₅₀ X₉₀ VMD GSD (<5.8 μm)FDKP diammonium salt with 078 1.70 μm 4.10 μm 8.40 μm 4.68 μm 1.8672.13% 25% insulin (w:w)

Particle size of a preparation of the diammonium salt of FDKP containing25% insulin (w:w) was determined by laser diffraction and demonstrated asize range of approximately 1.7 μm-8.4 μm for the FDKP ammonium saltformulated with 25% insulin (FIG. 7 and Table 7).

TABLE 7 Fine Particle Fraction Lot# Run X₁₀ X₅₀ X₉₀ VMD GSD (<5.8 μm)FDKP diammonium salt with 076 1.57 μm 4.51 μm 8.79 μm 4.97 μm 1.9166.95% 50% insulin (w:w)

Particle size of a preparation of the diammonium salt of FDKP containing50% insulin (w:w) was determined by laser diffraction and demonstrated asize range of approximately 1.6 μm-8.8 μm for the FDKP ammonium saltformulated with 50% insulin (Table 8).

TABLE 8 Fine Particle Fraction Lot# Run X₁₀ X₅₀ X₉₀ VMD GSD (<5.8 μm)SDKP diammonium salt with 084 1.66 μm 4.64 μm 9.27 μm 5.17 μm 1.9264.69% 25% insulin (w:w)

Particle size for the SDKP diammonium salt formulated with 25% insulin(w:w) was determined by laser diffraction and demonstrated a size rangeof approximately 1.7 μm-9.3 μm for the SDKP diammonium salt formulatedwith 25% insulin.

Scanning electron microscopy was utilized to study particle morphology.Representative SEMs are shown in the FIG. 10 (FDKP) and FIG. 11 (SDKP).The particle morphology is consistent with a collapsed hollow sphere.

The stability of the in situ salt formation and formulation of thediammonium salt/insulin particles was evaluated under accelerated roomtemperature conditions (40%/75% RH). Compared to a control formulationprepared by lyophilization, the spray dried particles demonstratedsuperior insulin stability as measured by insulin degradation (FDKP,FIG. 12 and SDKP, FIG. 14) and formation of the desamino degradrant(A₂₁) (FDKP, FIG. 13 and SDKP, FIG. 15).

Example 16 Characteristics of Spray Dried Microparticles

Spray dried FDKP salt/insulin particles demonstrate a surprising andunexpected trend in aerodynamic performance. Previously observedinsulin-containing microparticles, which had been formed from DKP freeacid microparticles onto which insulin had been loaded and the solventremoved by lyophilization, demonstrated decreased aerodynamicperformance with increasing insulin content. For example, the % rf(respirable fraction) for 25% loaded particles was significantly lowerthan the % rf for 5% loaded particles. For spray dried FDKP saltmicroparticles containing insulin, the opposite trend is observed. Asinsulin load increases, % rf increases.

Spray dried powders of the FDKP disodium salt were prepared with insulincontents of 11.4%, 50.0%, 70.0%, or 90.0% (w:w). FIG. 16 shows that % rfincreases with increasing insulin load.

A similar trend was also observed in spray dried FDKP diammoniumsalt/insulin powders having insulin contents of 11.4%, 50.0%, 70.0%, or90.0% (w:w). The % rf increased with insulin load (FIG. 17).

The starting concentration of the FDKP disodium salt solution prior tospray drying was evaluated for its effect on final particle insulinstability. The data indicate that insulin stability in the powderincreases with increasing solution concentrations as measured by insulinloss after 17 days at 40°/75% RH. For example, 8.5% insulin was lostfrom powder spray dried from a solution containing 37 mg/mL solids. Bycomparison, 4.5% insulin was lost from powder spray dried from asolution containing 45 mg/mL solids and 2.7% insulin was lost frompowder spray dried from a solution containing 67 mg/mL solids.

The inlet temperatures used to spray dry solutions of the FDKP disodiumsalt and insulin to form particles containing 50% insulin was evaluatedfor its effect on final particle insulin stability. The data indicatethat insulin stability in the powder increases with increasing inlettemperature as measured by insulin loss after 17 days at 40°/75% RH. Forexample, about 4% insulin was lost from powder spray dried at an inlettemperature of 180° C. By comparison, <1% insulin was lost from powderspray dried at an inlet temperature of 200° C.

Additionally, the present inventors have unexpected found that theseparticles, which are suitable for pulmonary delivery, have a rugosity ofapproximately 1.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar references used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of any and all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed is:
 1. A method of preparing a solid composition fordrug delivery comprising: preparing a solution containing a biologicallyactive agent and a pharmaceutically-acceptable salt of a heterocycliccompound in a solvent; and a step for removing said solvent; whereinsaid pharmaceutically acceptable salt of a heterocyclic compound has thestructure according to Formula 1:

wherein R₁ or R₂ are independently selected from succinate-4-aminobutyl,glutarate-4-aminobutyl, maleate-4-aminobutyl, citraconate-4-aminobutyl,malonate-4-aminobutyl, oxalate-4-aminobutyl, and fumarate-4-aminobutyl;E₁ and E₂ are NH₂; and said salt further comprises at least one cation.2. The method of claim 1, wherein said step for removing said solventcomprises distillation.
 3. The method of claim 1, wherein said step forremoving said solvent comprises evaporation.
 4. The method of claim 1,wherein said step for removing said solvent comprises spray drying. 5.The method of claim 1, wherein said step for removing said solventcomprises lyophilization.
 6. The method of claim 1, wherein said atleast one cation is selected from the group consisting of sodium,potassium, calcium, magnesium, lithium, triethylamine, butylamine,diethanolamine, and triethanolamine.
 7. The method of claim 1, whereinsaid at least one cation is sodium.
 8. The method of claim 1, whereinsaid biologically active agent is selected from the group consisting ofhormones, anticoagulants, immunomodulating agents, cytotoxic agents,antibiotics, antivirals, antisense, anti-inflammatories, vasoactiveagents, neuroactive agents, cannabinoids, antigens, antibodies andactive fragments and analogues thereof.
 9. The method of claim 1 furthercomprising the step of micronizing said solid to form a dry powder. 10.The method of claim 9, wherein the particles of said dry powder aresuitable for pulmonary delivery.
 11. The method of claim 9, wherein theparticles of said dry powder have a rugosity of less than
 2. 12. Themethod of claim 1, wherein said solid composition comprisesmicroparticles.
 13. The method of claim 12, wherein at least 50% of saidparticles have a diameter less than 5 μm.
 14. The method of claim 12,wherein at least 70% of said particles have a diameter less than 5 μm.15. diameter The method of claim 12, wherein said microparticles have arugosity of less than
 2. 16. The method of claim 12, wherein saidmicroparticles comprise a dry powder.
 17. The method of claim 12,wherein said microparticles are suitable for pulmonary delivery.
 18. Themethod of claim 1, wherein said solid composition is a precipitate. 19.The method of claim 1, wherein said solid composition is formulated intoa solid dosage form.
 20. The method of claim 19, wherein said soliddosage form is selected from the group consisting of tablets andcapsules.